Agrobacterium rhizogenes-Mediated Transformation of the Parasitic Plant Phtheirospermum japonicum

Background: Plants within the Orobanchaceae are an agriculturally important group of parasites that attack economically important crops to obtain water and nutrients from their hosts. Despite their agricultural importance, molecular mechanisms of the parasitism are poorly understood. Methodology/Principal Findings: We developed transient and stable transformation systems for Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae. The transformation protocol was established by a combination of sonication and acetosyringone treatments using the hairy-root-inducing bacterium, Agrobacterium rhizogenes and young seedlings. Transgenic hairy roots of P. japonicum were obtained from cotyledons 2 to 3 weeks after A. rhizogenes inoculation. The presence and the expression of transgenes in P. japonicum were verified by genomic PCR, Southern blot and RT-PCR methods. Transgenic roots derived from A. rhizogenes-mediated transformation were able to develop haustoria on rice and maize roots. Transgenic roots also formed apparently competent haustoria in response to 2,6dimethoxy-1,4-benzoquinone (DMBQ), a haustorium-inducing chemical. Using this system, we introduced a reporter gene with a Cyclin B1 promoter into P. japonicum, and visualized cell division during haustorium formation. Conclusions: We provide an easy and efficient method for hairy-root transformation of P. japonicum. Transgenic marker analysis revealed that cell divisions during haustorium development occur 24 h after DMBQ treatment. The protocol

Cigarette Smoke Affects Keratinocytes SRB1 Expression and Localization via H2O2 Production and HNE Protein Adducts Formation

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H2O2) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H2O2, induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake

MOESM4 of Factors affecting the efficiency of Rhizobium rhizogenes root transformation of the root parasitic plant Triphysaria versicolor and its host Arabidopsis thaliana

Additional file 4: Table S3. Sequences of primers used for PCR

Chemical induction of hairpin RNAi molecules to silence vital genes in plant roots

Understanding the functions encoded by plant genes can be facilitated by reducing transcript levels by hairpin RNA (hpRNA) mediated silencing. A bottleneck to this technology occurs when a gene encodes a phenotype that is necessary for cell viability and silencing the gene inhibits transformation. Here we compared the use of two chemically inducible plant promoter systems to drive hpRNA mediated gene silencing in transgenic, hairy roots. We cloned the gene encoding the Yellow Fluorescence Protein (YFP) into the dexamethasone inducible vector pOpOff2 and into the estradiol induced vector pER8. We then cloned a hpRNA targeting YFP under the regulation of the inducible promoters, transformed Medicago truncatula roots, and quantified YFP fluorescence and mRNA levels. YFP fluorescence was normal in pOpOff2 transformed roots without dexamethasone but was reduced with dexamethasone treatment. Interestingly, dexamethasone removal did not reverse YFP inhibition. YFP expression in roots transformed with pER8 was low even in the absence of inducer. We used the dexamethasone system to silence acetyl-CoA carboxylase gene and observed prolific root growth when this construct was transformed into Medicago until dexamethasone was applied. Our study shows that dexamethasone inducibility can be useful to silence vital genes in transgenic roots

Applications of NDE to the Processing of Metals

Presently conceived automated metals processing systems have reached a high degree of complexity and incorporate not only control hardware but algorithms based on computer simulations and models of processes and a multiplicity of sensors for monitoring process and geometrical parameters, as well as material properties during the various stages of processing1-4. In such systems, sensors which can nondestructively measure material properties during processing provide information which can be used to verify, simplify and eventually improve the control algorithms. Also by directly providing the quantities of interest, such sensors relax the requirements on other measurements (such as temperature) from which material properties are traditionally inferred. In addition, material property sensors used near the end of the process insure that specifications are being met, regardless of the performance of automated systems upstream. In some cases the availability of new techniques capable of monitoring the evolution of microstructure during initial phases of processing may also help develop new and simpler metallurgical processes resulting in simultaneous improvements of quality and productivity